TCF25 as a Nutrient Sensor in Lysosomal Acidification During Glucose Starvation

Study Background and Research Question

Cells rely on glucose for energy and biosynthetic processes. Under glucose starvation, cells activate adaptive mechanisms such as AMP-activated protein kinase (AMPK) signaling and autophagy to maintain energy homeostasis. Lysosomes are central to these adaptations, mediating autophagic degradation and metabolic recycling. However, while short-term autophagy is protective, prolonged or excessive lysosomal activity may trigger lysosome-dependent cell death (LDCD), contributing to tissue injury in metabolic disorders. The molecular regulators that coordinate lysosomal adaptation and cell death during nutrient stress remain incompletely understood (Ren et al., 2025).

Key Innovation from the Reference Study

The study by Ren et al. (2025) introduces a genome-wide CRISPR-Cas9 screening approach to identify determinants of cell fate under glucose deprivation. The authors pinpoint transcription factor TCF25 as a previously unrecognized nutrient sensor that orchestrates metabolic adaptation by enhancing lysosomal acidification through V-type H⁺-ATPase (V-ATPase) activity. This TCF25-driven process is crucial not only for autophagy-mediated survival under transient stress but also accelerates ferritinophagy and LDCD during sustained glucose starvation (Ren et al., 2025).

Methods and Experimental Design Insights

The investigators conducted a genome-wide CRISPR-Cas9 knockout screen in cultured cells subjected to glucose starvation to identify genes essential for cell death under metabolic stress. Enrichment analysis revealed a significant representation of lysosomal pathway genes, focusing attention on TCF25. Subsequent experiments involved TCF25 knockout and overexpression, assessment of lysosomal pH, autophagic flux, and V-ATPase function. The authors also employed mouse models of hepatic ischemia-reperfusion injury to translate their findings in vivo (Ren et al., 2025).

Protocol Parameters

• assay | CRISPR-Cas9 genome-wide screen | variable (depends on library and cell line) | identifies essential genes for glucose-starvation-induced cell death | paper
• assay | glucose starvation | 0 mM glucose for 24–48 h | models metabolic stress and lysosomal adaptation | paper
• assay | lysosomal acidification measurement | LysoSensor dye, flow cytometry | quantifies V-ATPase-dependent pH changes | paper
• assay | TCF25 knockout or overexpression | genetic manipulation | validates gene function in adaptation and death | paper
• assay | V-ATPase inhibition (e.g., Concanamycin A) | 20 nM, 60 min | blocks lysosomal acidification for mechanistic dissection | product_spec
• assay | ferritinophagy and LMP assays | immunoblotting, microscopy | links TCF25 activity to lysosomal membrane integrity | paper
• assay | mouse hepatic ischemia-reperfusion model | TCF25 knockout vs. wildtype | tests physiological relevance of findings | paper

Core Findings and Why They Matter

Ren et al. demonstrate that TCF25 is upregulated in response to glucose deprivation and localizes to lysosomes, where it modulates V-ATPase assembly and activity. Enhanced V-ATPase function lowers lysosomal pH, promoting autophagic flux and energy maintenance during early nutrient stress. However, with prolonged starvation, TCF25-driven acidification accelerates ferritinophagy—selective degradation of ferritin—culminating in increased lysosomal membrane permeability and cell death. Notably, genetic ablation of TCF25 or pharmacological inhibition of V-ATPase (e.g., with Concanamycin A) prevents lysosomal acidification, autophagic flux, and LDCD, highlighting the dual role of TCF25 in survival and death pathways (Ren et al., 2025).

In mouse models, TCF25 deficiency conferred resistance to hepatic ischemia-reperfusion injury, underscoring the translational potential of modulating lysosomal acidification in metabolic diseases. These findings position TCF25 as a metabolic sensor that integrates nutrient cues to regulate autophagy, ferritinophagy, and cell fate through lysosomal pH control.

Comparison with Existing Internal Articles

Several internal resources, such as "Reengineering Tumor Cell Fate: Strategic Advances with Concanamycin A", highlight the use of Concanamycin A as a gold-standard V-type H⁺-ATPase inhibitor to disrupt endosomal acidification and induce apoptosis in cancer models. These articles focus on the mechanistic disruption of pH homeostasis and the induction of apoptosis in tumor cells via V-ATPase inhibition (internal resource). The present study extends this mechanistic framework by showing that V-ATPase activity, regulated by TCF25, is not only crucial in cancer but also in the metabolic adaptation and cell death of non-malignant cells under nutrient stress. The convergence of findings across tumor and non-tumor systems underscores the centrality of V-ATPase-mediated lysosomal acidification in diverse cell fate decisions, supporting broader applications for selective V-ATPase inhibitors in both cancer biology research and metabolic disease models.

Limitations and Transferability

While the study offers robust evidence for the role of TCF25 and V-ATPase in lysosomal acidification and cell fate during glucose starvation, several limitations should be noted. First, the cell culture and mouse models used may not capture the full complexity of human pathophysiology. Second, while pharmacological inhibition with agents like Concanamycin A supports the mechanistic claims, off-target effects and differences between acute and chronic inhibition were not fully addressed (Ren et al., 2025; workflow_recommendation). Finally, the relationship between TCF25 and other metabolic sensors warrants further exploration, particularly in tissues beyond the liver and in disease settings beyond ischemia-reperfusion injury.

Research Support Resources

For researchers aiming to dissect V-ATPase-dependent lysosomal acidification and related cell fate pathways, Concanamycin A (SKU A8633) offers a potent and selective means to inhibit V-ATPase, disrupt endosomal acidification, and model apoptosis or cytoprotection in both cancer and non-cancer systems (source: product_spec). APExBIO's Concanamycin A is widely used in protocols involving apoptosis induction in tumor cells and inhibition of endosomal acidification, with recommended treatment conditions of 20 nM for 60 minutes in a variety of cell lines (source: product_spec). This reagent can thus enable rigorous modeling of the mechanisms delineated by Ren et al. and facilitate further investigation into lysosomal regulation under metabolic stress.